Antibody binding a linear epitope of human p53 and diagnostic applications thereof

ABSTRACT

The invention relates to an anti-human p53 antibody suitable for specifically binding a linear epitope which is exposed only in a conformationally altered isoform of the characteristic p53 protein of patients with Alzheimer&#39;s disease or prone to develop Alzheimer&#39;s disease or cognitive impairment during ageing. Methods and diagnostic and prognostic kits are also described.

CROSS REFERENCE TO RELATED APPLICATION

The present application is a continuation of and claims priority to U.S.patent application Ser. No. 17/093,541, filed Nov. 9, 2020, and entitled“Antibody Binding a Linear Epitope of Human p53 and DiagnosticApplications Thereof,” which is a continuation of and claims priority toU.S. patent application Ser. No. 16/214,455, filed Dec. 10, 2018, andentitled “Antibody Binding a Linear Epitope of Human p53 and DiagnosticApplications Thereof,” and which issued as U.S. Pat. No. 10,875,908 onDec. 29, 2020, which is a continuation of and claims priority to U.S.patent application Ser. No. 15/515,693, filed Mar. 30, 2017, andentitled “Antibody Binding a Linear Epitope of Human p53 and DiagnosticApplications Thereof,” and which issued as U.S. Pat. No. 10,183,990 onJan. 22, 2019, which is a U.S. national stage of and claims priority toInternational Patent Application No. PCT/EP2015/072094, filed Sep. 25,2015, which claims priority to and the benefit of Italian PatentApplication No. TO2014A000776, filed Sep. 30, 2014, the contents of eachof which are herein incorporated by reference in their entirety.

SEQUENCE LISTING

This application contains a Sequence Listing which has been submittedelectronically in ASCII format and is hereby incorporated by referencein its entirety. Said ASCII copy, created on Sep. 30, 2021, is named121689-10105-seq_listing.txt and is 14 kilobytes in size.

BACKGROUND OF THE INVENTION

The present invention lies in the field of immunodiagnostics.

More particularly, the present invention relates to an antibody, a kitand in vitro methods of diagnosing Alzheimer's disease and/or thepredisposition of a subject to develop Alzheimer's disease or cognitiveimpairment during ageing.

The DNA binding domain (DBD) of human p53 protein (aa 101-306) ischaracterized by high-grade conformational flexibility and contains oneZn²⁺ ion coordinated by three cysteines (residues 176, 238 and 242) andone histidine (residue 179). In the wild-type isoform of human p53protein, the aa 282-297 linear epitope is masked and therefore notavailable for recognition by an epitope-specific antibody.

However, because of the high-grade flexibility of its DBD domain, p53protein can assume several conformations, which also characterize itsbiological activity.

In its wild-type conformation, p53 can bind DNA consensus sequences andtranscribe/repress the expression of target genes. In this conformation,the protein exposes an epitope which is recognized by the commerciallyavailable, conformationally specific antibody named PAb1620.

There are several grades of conformational alteration states of p53,which correspond to different conformationally altered isoforms. Somemutations in the p53 gene cause a conformational change of the protein,which corresponds to a conformationally altered isoform capable of beingrecognized by commercially available, conformationally specificantibodies.

Additional conformationally altered isoforms may also derive frompost-translational modifications, such as oxidation and/or nitrationreactions, which alter the wild-type tertiary structure of the protein.

Buizza L. et al. (“Conformational altered p53 as an early marker ofoxidative stress in Alzheimer's disease”, PlosOne 7(1):e29789) andUberti D et al. (“Identification of a mutant-like conformation of p53 infibroblasts from sporadic Alzheimer's disease patients”, Neurobiology ofAging 27 (2006) 1193-1201) both identify the expression of mutated p53protein in patients with Alzheimer's disease, such identification havingbeen made using the anti-p53 antibody PAb240 which binds a crypticepitope on the amino acid residues 213-217 of p53 protein, notdetectable in the wild-type p53 protein.

BRIEF SUMMARY OF THE INVENTION

The present inventors have unexpectedly found an anti-human p53 whichhas proved capable of specifically and selectively recognize aconformationally altered isoform of the protein resulting from itspost-translational modifications, in which the aa 282-297 linear epitopeis exposed.

The inventors have also unexpectedly observed that the isoform of p53protein recognized by the antibody of the invention is peculiarlyexpressed in subjects with Alzheimer's disease. In particular, theconformationally altered isoform recognized by the antibody of theinvention is expressed in higher amounts in biologicalsamples—particularly in samples of blood cells, neuronal cells or othercell types, as well as in samples of biological fluids such as, forexample, blood, plasma, serum, saliva, urine—of patients withAlzheimer's disease.

The inventors further observed that such conformationally alteredisoform recognized by the antibody of the invention is also expressed inbiological samples of subjects with Mild Cognitive Impairment (MCI).

Therefore, the antibody of the invention is a useful diagnostic andprognostic tool, for diagnosing Alzheimer's disease and for determiningthe predisposition of a subject with Mild Cognitive Impairment (MCI) todevelop Alzheimer's disease.

Finally, the inventors observed that the expression of the p53 proteinisoform positive to the antibody of the invention in a statisticallysignificant manner correlates with the age and cognitive impairment ofthe subjects studied.

Therefore, the antibody of the invention is also useful for determiningthe predisposition of a subject to develop cognitive impairment duringageing.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 discloses a P53 protein sequence;

FIG. 2 discloses experimental data of 2d3A8/1620 ratio versus CONT, SADand FAD;

FIG. 3 discloses experimental data of example 4 of unfolded P53 PBMC2d3A8 absorbance versus CONT, MCI and AD;

FIG. 4 discloses experimental data of example 5, during ageing, ofunfolded P53 PBMC 2d3A8 absorbance versus CONT, MCI and AD; and

FIG. 5 discloses experimental data of example 5, decreasing of thescoring in the MMSE test, of unfolded P53 PBMC 2d3A8 absorbance versusCONT, MCI and AD.

DETAILED DESCRIPTION OF THE INVENTION

A first object of the present invention is therefore an anti-human p53antibody, characterized in that it recognizes the linear epitope ofsequence RRTEEENLRKKGEPHH (SEQ ID NO:1) present in the DNA bindingdomain (DBD) of human p53, said linear epitope spanning between theamino acid positions 282-297 of the amino acid sequence of human p53.

The preparation of the antibody of the invention is described in thefollowing experimental part.

In a preferred embodiment, the antibody of the invention is a monoclonalantibody.

The antibody of the present invention can be obtained by any well-knownmethodology for the preparation of polyclonal or monoclonal antibodies.In the following experimental part, the preparation of the antibody byanimal (mice) immunization with an antigen consisting of a peptide ofsequence CRTEEENLRKKGEPHH (SEQ ID NO:2) conjugated with bovine serumalbumin as carrier and hybridoma technique is described by way ofexample.

As previously mentioned, the antibody object of the present inventionspecifically recognizes an isoform of human p53 protein which has beenshown to be correlated to Alzheimer's disease and development ofcognitive impairment during ageing. Such antibody therefore represents auseful diagnostic and prognostic tool.

An in vitro method of determining an isoform conformationally altered bypost-translational modification of p53 protein peculiar of Alzheimer'sdisease, as well as the diagnostic and prognostic methods as defined inthe appended claims which form an integral part of the presentdescription, are also part of the present invention.

The method detects the formation of an immunocomplex between the humanp53 protein and the antibody of the sample.

An immunodiagnostic kit as defined in the appended claims is also partof the present invention.

In order to implement the methods and kit of the invention any type ofwell-known immunoassay can be used, such as for exampleimmunoprecipitation assay, ELISA or RIA, immunofluorescence, WesternBlot, FACS analysis, immunocytochemistry/immunohistochemistry.

An immunoassay kit may include the antibody and means for detecting thebinding of the antibody to human p53 protein.

The following non-limitative examples are provided to illustrate thescope of the invention as defined by the appended claims.

Example 1

1a. Immunization

For the immunization, 6/8 week old mice which were healthy anddisorder-free were used. The peptide used as antigen for antibodyproduction had the following features:

-   -   Sequence: “N-terminal” CRTEEENLRKKGEPHH “C terminal” (SEQ ID NO:        2)    -   Length: 16 amino acids    -   Molecular weight: 1960.94    -   Purity: 96.4%    -   Form: lyophilized powder    -   Conjugation: Conjugated with BSA by the glutaraldehyde method

The p53 protein sequence (SEQ ID NO: 3) is depicted in FIG. 1, where theDNA binding domain is highlighted in grey, and the linear epitoperecognized by 2D3A8 antibody is underlined. The amino acid sequences ofthe 2D3A8 antibody include the heavy chain (SEQ ID NO: 4) and lightchain (SEQ ID NO: 5), heavy chain variable region (SEQ ID NO: 6) andlight chain variable region (SEQ ID NO: 7), heavy chain CDRs 1, 2 and 3(SEQ ID NOS: 8, 9 and 10, respectively) and light chain CDRs 1, 2 and 3(SEQ ID NOS: 11, 12 and 13, respectively).

The first injection has been performed by emulsifying the antigen (50μg) in Freund's complete adjuvant (FCA). The subcutaneous injectionshave been performed in 2-3 sites on the animal. Additional injectionshave been performed at 3-week intervals with 50 μg of antigen emulsifiedin Freund's incomplete adjuvant (FIA). The antibody titer is evaluatedby ELISA.

In the ELISA assay, the antibody titer present in the serum of 5 micewas evaluated after the third injection with the above-describedpeptide. The blood of immunized mice was collected from their caudalveins. The absorbance values obtained following the spectrophotometricreading provided important information on the antibody titer present inthe various mice. Animals were subjected to further additionalinjections so that the antibody titer reached a sufficiently high level.The mouse with the best antibody titer was chosen for the first fusion.

1b. Development of Hybridomas

The animals' splenocytes were fused with mouse myeloma cells (SP2/O cellline). The fusion products were subjected to screening against theantigen to select the antibody-producing clones. The growth of theseclones was continued. This first screening was performed by ELISAmethodology. The positive clones were labeled as “parental clones” andfrozen after 3 passages. An antigen coating was created onto ELISAplates, and successively the fusion product supernatant was added. Theserum of the immunized animals was used as a positive control in ELISA(Example 2).

Example 2

After the fusion between the splenocytes of the mouse with the bestantibody titer and mouse myeloma cells, an ELISA assay was performed toevaluate the fusion products. A coating with the antigen was created ina 96-well ELISA plate and serial dilutions of the supernatant fromvarious clones were added to each well to evaluate their antibodyproduction by spectrophotometric reading. The clones with the highestoptical density at 450 nm (OD_(450 nm)) were transferred to 24-wellplates and after their growth the ELISA assay was repeated, the cloneswith the highest antibody production were transferred to 6-well plates,grown and tested again by ELISA. The procedure was repeated also for theclones transferred to culture flasks. These successive assays allowedthe identification of the best clones which were tested for the lasttime by ELISA, with the limit dilution method to ensure that thepositive clones showed an actual antibody response.

The validated antibody was purified from the supernatant of the clonewith the highest OD_(450 nm) value and therefore with the best antibodytiter. This antibody is named “clone 2D3A8” for brevity.

Example 3

Study of the expression of the conformationally altered isoform of p53protein recognized by 2D3A8 antibody in patients with sporadic andfamilial Alzheimer's disease and MCI.

In immortalized B lymphocytes of patients diagnosed with sporadicAlzheimer (SAD) and familial Alzheimer (FAD), the conformational stateof p53 was evaluated by the immunoprecipitation method, using twoconformationally specific antibodies which recognize the wild-typeisoform of the protein (PAb 1620) and a conformationally altered isoform(2D3A8). The immunoprecipitate was then visualized by Western Blot witha polyclonal anti-p53 antibody (CM1). The experimental data wereexpressed as ratio between the intensity of the band positive to 2D3A8antibody and to PAb1620 of the same sample.

In SAD and FAD samples, the 2D3A8/1620 ratio was significantly highercompared to the lymphocytes of dementia-free control patients (FIG. 2).

2D3A8 antibody can therefore discriminate a conformationally alteredisoform of p53 peculiarly expressed in immortalized lymphocytes ofpatients with sporadic (SAD) and familial (FAD) Alzheimer.

Example 4

In samples of fresh blood of patients diagnosed Alzheimer and ofsubjects with mild cognitive impairment, diagnosed MCI, theconformationally altered isoform of p53 recognized by 2D3A8 antibody(2D3A8-positive p53) was evaluated by ELISA. Healthy, dementia-freesubjects of the same age were also evaluated.

2D3A8-positive p53 vas detected both in blood cells (PBMC) and in serumof the same patients or subjects. 2D3A8 antibody can recognize withhigh-specificity grade patients with Alzheimer. Interestingly, subjectswith mild cognitive impairment express serum levels of 2D3A8-positivep53 statistically higher than the levels of protein isoform present incontrol subjects. In PBMCs and in serum of patients with Alzheimer, the2D3A8-positive p53 isoform was statistically increased compared to thecontrols (FIG. 3).

Example 5

The 2D3A8-positive p53 isoform correlates with age.

During ageing, the expression of 2D3A8-positive p53 in blood cells(PBMC) increases in a statistically significant manner (FIG. 4).

Moreover, the expression of 2D3A8-positive p53 correlates with thecognitive status, measured by the well-known neuropsychological testMMSE. The 2D3A8-positive p53 isoform increases with the decrease of thescoring obtained in the MMSE test, i.e., it increases with theprogression of cognitive impairment (FIG. 5).

1. A method of identifying in a subject the presence of Alzheimer'sdisease which is sporadic Alzheimer's disease (SAD) or familialAlzheimer's disease (FAD) comprising the steps of (i) contacting abiological sample of said subject with a) an antibody specific for analtered isoform of human p53 protein, said antibody comprising a heavychain variable region comprising complementary determining region (CDR)1 (SEQ ID NO:8), CDR2 (SEQ ID NO: 9) and CDR3 (SEQ ID NO: 10) and alight chain variable region comprising CDR1 (SEQ ID NO:11), CDR2 (SEQ IDNO: 12) and CDR3 (SEQ ID NO: 13) and b) an antibody specific for a wildtype conformation of human p53 protein, wherein said contacting isperformed under conditions which permit formation of an immunocomplexbetween said antibody and said human p53 protein isoform; (ii)quantifying the amount of immunocomplex formed between said alteredisoform of human p53 protein and said antibody specific for said alteredisoform; (iii) quantifying the amount of immunocomplex formed between awild type isoform of human p53 protein and an antibody specific for saidwild type human p53 protein isoform (iv) determining a ratio of saidamount of quantified immunocomplex of (ii) and said amount of quantifiedimmunocomplex of (iii) in said sample, wherein a ratio greater than 1indicates the presence of sporadic (SAD) or familial (FAD) Alzheimer'sdisease in said subject.
 2. The method of claim 1, wherein the firstantibody comprises antibody 2A3D8.
 3. The method of claim 1, whereinsaid antibody is monoclonal.
 4. The method according to claim 1, whereinsaid heavy chain variable region comprises SEQ ID NO: 6 and said lightchain variable region comprises SEQ ID NO:
 7. 5. The method according toclaim 1, wherein said biological sample comprises lymphocytes, plasma,serum or blood.
 6. The method according to claim 1, wherein saiddetection comprises one or more of immunoprecipitation, ELISA, RIA,immunofluorescence, Western Blot, FACS immunocytochemistry and,immunohistochemistry.
 7. The method according to claim 1, wherein theantibody comprises a heavy chain and a light chain.
 8. A method ofidentifying Alzheimer's Disease (AD) in a subject, comprising (i)contacting a biological sample of said subject with an antibody specificfor an altered isoform of human p53 protein, said antibody comprising aheavy chain variable region comprising complementary determining region(CDR) 1 (SEQ ID NO:8), CDR2 (SEQ ID NO: 9) and CDR3 (SEQ ID NO: 10) anda light chain variable region comprising CDR1 (SEQ ID NO:11), CDR2 (SEQID NO: 12) and CDR3 (SEQ ID NO: 13); (ii) quantifying the amount of animmunocomplex formed between said altered isoform of human p53 proteinand said antibody, wherein the presence of a higher amount of saidimmunocomplex when compared with the amount of said immunocomplex foundin a dementia-free control patient identifies Alzheimer's disease insaid subject.
 9. The method of claim 8, wherein said heavy chainvariable region comprises SEQ ID NO: 6 and said light chain variableregion comprises SEQ ID NO:
 7. 10. The method of claim 8, wherein saidbiological sample comprises plasma.
 11. The method of claim 10, whereinsaid sample comprises blood.
 12. The method of claim 8, wherein saiddetection comprises one or more of immunoprecipitation, ELISA, RIA,immunofluorescence, Western Blot, FACS, immunocytochemistry, andimmunohistochemistry.
 13. A method of detecting an isoform of human p53protein in a biological sample of a human subject, said isoform beingconformationally altered with respect to the wild-type human p53protein, the method comprising the steps of: a. contacting the samplewith an anti-human p53 antibody comprising a heavy chain variable regioncomprising CDR1 (SEQ ID NO:8), CDR2 (SEQ ID NO: 9) and CDR3 (SEQ ID NO:10) and a light chain variable region comprising CDR1 (SEQ ID NO:11),CDR2 (SEQ ID NO: 12) and CDR3 (SEQ ID NO: 13), and b. detectingformation of an immunocomplex between human p53 protein and saidantibody in said sample, wherein said formation of said immunocomplexindicates the presence of said isoform in said sample.
 14. The methodaccording to claim 13, wherein said heavy chain variable regioncomprises SEQ ID NO: 6 and said light chain variable region comprisesSEQ ID NO:
 7. 15. The method according to claim 13, wherein saidbiological sample comprises plasma.
 16. The method according to claim15, wherein said sample comprises blood.
 17. The method according toclaim 13, wherein said detection comprises one or more ofimmunoprecipitation, ELISA, RIA, immunofluorescence, Western Blot, FACSimmunocytochemistry and, immunohistochemistry.
 18. The method accordingto claim 13, wherein the antibody comprises a heavy chain and a lightchain.
 19. The method according to claim 13, wherein said antibody is amonoclonal antibody.